light-induced water splitting and hydrogen production in ... · light-induced water splitting and...

Alternative Energy Seminar,

Weizmann Institute, Rehovot, Israel – 24 February 2013

Wolfgang Lubitz

Light-Induced Water Splitting and Hydrogen

Production in Nature:

Blueprints for the Design of Chemical Catalysts

Max Planck Institute for Chemical Energy Conversion

Mülheim/Ruhr, Germany

2 H2O

Hydrogenase H2 production

Mn

+

Mn Mn

Mn

Ca

hn

e-

"Biomimetics"

Energy Storage

N2

Biomass/Food

Rubisco

CO2 reduction

ATP NADPH

Solar Fuel

4 H+ + O2 2 H2O O2, 4 H+

Solar Fuel

4H+

hn

CO2 CO2

2H2

NH3 CH4 CH3OH

Molecular Concepts of Water Splitting: Nature’s Approach

Oxygenic Photosynthesis Biomimetic System

2 H2O

O2

Fuel cell

+ + + +

Chemical Energy Storage - Solar Fuel Production

PS II

Nicholas Cox, Wolfgang Lubitz, MPI for Chemical Energy Conversion, Mülheim/Ruhr

Karamanolis: Wasserstoff, Energieträger der Zukunft, 2001, Elektra Verlag

Hydrogen: Energy Carrier of the Future Basic material for production of other energy carriers (e. g. methanol)

other chemicals and other uses (e. g. ammonia)

Energy carrier

Electronics

Metallurgy

Refrigeration

Welding technology

Artificial production

of diamonds Glass industry

Production of

methanol Production of

Ammonia (Haber Bosch process)

Steato

chemistry

Food industry

Balloon gas

Hydrogen

Utilization

1. Hydrocarbons

Reformation of natural gas, coal

2. Water electrolysis

Electricity from nuclear power or fossil resources

Renewable energy (photovoltaics, wind ….)

3. Thermal methods

Waste heat (nuclear power)

Solar-Tower

4. Biological / Biomimetic Methods

A. Modified natural systems (biophotolysis / photosynthesis)

B. Semiartificial systems (enzymes on electrodes)

C. Biomimetic systems (artificial photosynthesis, catalysis)

HYDROGEN PRODUCTION

Photosynthesis:

Structure and Function of Photosystem II

and the Water Oxidizing Complex

water

+

carbon

dioxide

oxygen +

carbo-

hydrates

fossil fuels

oil, coal, gas

food

renewable raw materials

biomass

H2O + CO2 (CH2O) + O2

carbohydrates chlorophyll

light

oxygen atmosphere

ozone layer

sunlight

Oxygenic Photosynthesis

section of a leaf chloroplast

grana O2

CO2

Photosynthesis: Light & Dark Reactions

thylakoid membrane

H2O

Photosynthetic

membrane

NADPH ATP

ADP+Pi

oscp

CF1

½ H2O 1/4 O2+

PS II PS I ATP synthase Cyt b6f

CF0

d F6

Antenna

(Lhcb)

Stroma

Membrane

hn

hn

dark reactions

Calvin cycle

CO2 CO2-fixation to

carbohydrates (CH2O)

enzyme Rubisco

Em (V)

1.2

0.8

0.4

0

-0.4

-0.8

-1.2

Fx

FA / FB Fd

YZ

Mn(Si)

P680

FNR

FeS Cyt f PC

QA

PQ

QB

A0

A1

cyt b6/f

complex

1-3 ps

30 ps

200 ns

800 ns

50 - 1200 µs

20-250 ns

300 ps

300 µs

~ 5 ms

~ 1 ms

cyt b6H

cyt b6L

< 300 µs

< 1-125 µs

< 1 ms

2 H2O

O2 + 4 H+

½ NADPH

½ H+ ½ NADP+ +

Pheo

P680

P700

2H+

2H+

3 ps

Z

Z

90°

P700

Electron Transport in Oxygenic Photosynthesis

Z-Scheme

local pseudo-C2 axis

X-Ray Crystallography: Structure of Photosystem II

Arrangement of water molecules

Umena, Kawakami, Shen, Kamiya, Nature 473, 55-60 (2011) Resolution 1.9 Å

cytoplasm

lumen T. vulcanus

CP43 CP47 D1 D2

lumen PsbO PsbU

PsbV

cytoplasm

C2-Axis

cyt b-559

1300 water molecules

in one monomer

20 subunits 35 Chlorophylls

2 Pheophytins

11 Carotens

>20 lipids

2 plastoquinones

1 Fe, 2 hemes

Mn4Ca-cluster

X-Ray Crystallography: Structure of Photosystem II

Cofactor Arrangement : Reaction Center

Resolution 1.9 Å

local pseudo-C2 axis

ChlD2

PheoD2

QB

CarD2 ChlzD2

Cyt b-559

cytoplasm

lumen

TyrD

Fe

P680

QA

PD2

ChlD1

Mn4Ca Cluster

CarD1

ChlzD1

PheoD1

TyrZ

T. vulcanus

PD1

CP43 CP47 D1 D2

lumen PsbO PsbU

PsbV

cytoplasm

C2-Axis

cyt b-559

Mg

N N

N N

D

H

H3C

H

H3C A

H

H

H

H CH3

B CH2

CH3

H

CH3

O C O OCH3 COO-phytyl

H2C

CH2 H

C

E

H Chl a Umena, Kawakami, Shen, Kamiya,

Nature 473, 55-60 (2011)

Model Structures of the Mn4OxCa Cluster

MnA

MnD

MnC

MnB Ca

X-Ray-Crystallography

1.9 Å Resolution

(„little“ radiation damage)


3.5 Å Resolution

(radiation damage)

Ferreira et al., Science 303, 1831 (2004)

Gusko et al., Nat. Struct. Mol. Biol. (2009)


2.9 Å Resolution

(radiation damage)

2.6 Å

2.5 Å

2.4 Å

2.5 Å

2.7 Å

2.4 Å

Umena et al.,

Nature (2011)

Intact PSII

Dose

100 K

13.3 keV

10 K

Mn XANES of PS II Crystal

X-ray damage reduces the Mn4(III2IV2) cluster (S1 State) to Mn(II) and

alters the geometric and electronic structure significantly

Mn Models

Yachandra, Messinger et al. 2005 Yano et al. 2005 PNAS

102, 12047-12052

6540 6550 6560 6570 0.0

1.7

F / I

0

Energy (eV)

C B

A

6540 6550 6560 6570 0.0

1.7

F / I

0

Energy (eV)

Mn(II)

Mn2(IV,IV)

Mn2(III,III)

XANES and EXAFS at Different X-ray Doses

Photoreduction of higher metal oxidation states

Mn K-edge

XRD at 100K, 13.3 keV

3.5*1010 photons

XAS at 10K, 6.6keV

1*107photons

A: 25% Mn(II); 3% total dose

B: 45% Mn(II); 15% total dose

C: 75% Mn(II);~50% total dose Photons (*1010/µm2)

Intact PSII

Dose

100 K

13.3 keV

10 K

6540 6550 6560 6570 0.0

1.7

F / I

0 Energy (eV)

C

B

A

0 0.5 1.0 1.5 2.0 2.5 3.0 0

20

40

60

80

Mn(I

I) c

on

ten

t (%

)

Solution

100 K, 13.3keV

Crystal

100 K, 13.3keV

Crystal

10 K, 13.3keV

0 2 4 6 8

FT

Am

plitu

de

Apparent Distance R+D (Å)

A

B

C

Intact PSII

Mn Mn O

O

Mn Mn O

Mn Mn O

Mn O

Mn EXAFS

Radiation Damage in X-Ray Diffraction: Photoreduction

X-ray damage reduces the Mn4(III2IV2) cluster (S1 State) to Mn(II) and alters the

geometric and electronic structure significantly

Yano et al. 2005 PNAS 102, 12047-12052

diffraction

pattern

particle injection

100 nm focus

~1021 W/cm2

- biological nano-crystals

- virus structures, virus genomes

- non-crystallisable proteins: 60 %

Measure structure then destroy?

Laue lense

Imaging Biomolecules

Avoid radiation damage (even at RT)

R. Neutze et al, Nature 406 (2000)

Explore time resolution

Structure of short-lived intermediates

- 1012-13 photons

- 10 keV

- 10 fs pulse

Free Electron Laser

(A) Light microscope image of crystals (average size of 10 μm) of PS II

used for the XRD measurements. (B) X-ray diffraction pattern of PS II

obtained at CXI using a pulse width of <50 fs and a flux of 3.4 × 1011

photons∕pulse at 9 keV. Resolutions of some highlighted Bragg spots

are given in yellow and resolution at edges of the selected area of the

detector are indicated by white dashed circles. The background was

removed by subtracting the average image of 1,052 misses recorded

directly before and after the crystal diffraction. (C) Enlarged view of an

area in the top left corner of the diffraction pattern shown in B (marked

by a blue box) to show highest resolution spots observed.

PNAS (2012)

Comparison of electron density computed

from CXI data with SR data truncated to 6.5 Å resolution.

Overview of the electron density (blue mesh)

for one monomer of PS II computed from the CXI

data (A) and from the truncated SR data (B). Protein

is shown as cartoon in yellow; 2mFo-DFc electron

density is contoured at 1σ; view is along the membrane

plane with cytoplasm at top. Electron density

in the region of the Mn4CaO5 cluster computed from

the CXI data (C) or from the truncated SR data (D),

respectively, view direction is approximately 90 deg

rotated with respect to Fig. 2B and is along the membrane

plane, with cytoplasm at the top, lumen at the

bottom. Protein is shown as cartoon in yellow (D1)

and magenta (CP43), Mn as violet spheres and Ca

as orange sphere. The lumenal end of TMH c as well

as helix cd and the C-terminal helix (eC) of D1 and the

ef helix of CP43 are labeled. The 2mFo-DFc electron

density map is contoured at 1σ (blue mesh); the difference

density (mFo-DFc map) is shown at þ3σ

(green mesh) and at −3σ (red mesh).

Refinement of the OEC Structure

Ames et al., JACS (2011)

Pantazis et al. Angew. Chem. (2012) Umena et al., Nature (2011)

x-tal structure DFT-optimized structures

Upon geometry optimization O(5) relaxes to

form Mn-Mn µ-oxo bridge.

Now Mn-Mn and Mn-O distances comparable

with EXAFS.

1.87 3.16 O5 1.87

3.21

2.50

2.60 O5 O5

Seff = ½ Seff = 5/2

ground state 1 ground state 2

(+1 kcal/mol)

CP43-Glu354

Asp170

W1 W2

W3

W4

Ala344

Glu189

His332

Glu333

A4

B3

C2

D1

Ca

Cooperation Frank Neese

S2 :

4 Mn

1 Ca

5 oxygen bridges

4 water molecules W1 – W4

Protein: carboxylates and 1 His

Stroma

Lumen

CP43

33

12 or 18 c550 or 24

CP47

YZ

Mn4OxCa

YD

Pheo

D2

Pheo

D1 QA QB

P680

Fe

cyt b-559

Th

yla

ko

id M

em

bra

ne

Th

yla

ko

id M

em

bra

ne

+

+

Light

2 H2O → O2↑ + 4 H+ + 4 e-

4 hn

Photosystem II

2 H2O

4 H+

O2

2H2O O2+ 4H+

PS II - Functional Model

Activity Measurement:

O2 release via Clark Electrode

Functional Test:

O2 release after 1-4 µs-light flashes:

Joliot-Type Electrode




Functional Test:



induced by ms light flashes in spinach thylakoids

Flash number 3 7 11 15 19

pola

rogra

phic

sig

nal

Joliot et al., 1969 thylakoids

pH 6.3, 4° C

Messinger et al., 1993

S2

S0

S1

S3

S4

Kok, 1970

5 state cycle

1 2 3

4

H2O

H2O

O2

H+

H+

H+

H+

H2O

O2

Oxygen Evolution in PS II




Functional Test:



induced by ms light flashes in spinach thylakoids

Flash number 3 7 11 15 19

pola

rogra

phic

sig

nal

Joliot et al., 1969 thylakoids

pH 6.3, 4° C

Messinger et al., 1993

1 2 3

4

Oxygen Evolution in PS II

Kok, 1970

2H2O

H2O + OH

HO2

H2O2

e, H+

e, H+

e, H+

e, H+

O2

aqueous solution

0

1

2

3

4

5

Gib

bs e

nerg

y c

ha

ng

e /

eV

YZS0(2H2O)

YZS1

YZS2

YZS3

YZoxS3

YZS0(O2)coml.

YZS0 + O2

WOC

e, n2H+

e, n1H+

e, n0H+

e, n3H+

Water Oxidation in Aqueous Solution and in PS II:

Function of Water-Splitting Complex

Messinger & Renger, 2008

Stroma

Lumen

CP43

33

12 or 18 c550 or 24

CP47

YZ

Mn4OxCa

YD

Pheo

D2

Pheo

D1 QA QB

P680

Fe

cyt b-559

Th

yla

ko

id M

em

bra

ne

Th

yla

ko

id M

em

bra

ne

+

2 H2O → O2↑ + 4 H+ + 4 e-

4 hn

2H2O O2+ 4H+

Photosystem II

2 H2O

4 H+

O2

PS II and Water Oxidase - Problems & Nature´s Solutions

1. Light-induced single electron

transfer (ps) coupled to 4-electron

water oxidation process (ms)-

storage of oxidation equivalents

2. Redox leveling at the catalyst:

small and equal steps!

3. Electron-proton coupling at the

catalyst – charge balance

4. Interfacing: via a redox-active

tyrosine Yz

5. Water binding, orientation and

O-O bond formation –

catalysis mechanism

6. High oxidative power of P680+ -

protection

7. Chl triplets and singlet oxygen (D1

damage) - repair/replacement

Peter Nixon, 2010

Reassembly

mon. PS2

Sync. repl.

D1

CP47-RC

extrinsic SUs

mon. PS2

CP43

+ low MW SUs

Dim. PS2

Ligation of

cofactors

D1 Processing

Ribosome

PsbA mRNA

for new D1 insert

Assembly and Repair: Replacement of D1 Protein in PS II

D1 half lifetime in the light 30 min!

S0

S1 H+ e-

S2 S3

S4

H+

e-

H+ e-

H+ e- 200 250 300 350 400 450

Magnetic Field, mT

Messinger et al./Ahrling et al. 1997

S0

S2

Dismukes and Siderer, 1981

200 250 300 350 400 450

Magnetic Field, mT

ST = 1/2

ST = 1/2

(MnIII)3(MnIV)

(MnIII)2(MnIV)2

(MnIII)(MnIV)3

(MnIII)(MnIV)3

(MnIV)4 or

?

S-states differ by one

electron oxidation

H2O O2

H2O

Electronic Structure of the S-States: EPR

Mn(II) S=5/2

Mn(III) S=4/2

Mn(IV) S=3/2

I (55Mn)=5/2 → Hyperfine Structure

Metal Centers

and Radicals

in Proteins

PELDOR-DEER

distance between

electron spins

hfc of strongly

coupled nuclei

ELDOR-detected NMR Davies ENDOR

Mims ENDOR

hfc and nqc determination

Electron-Nuclear-Nuclear

Triple Resonance

signs and assignments

HYSCORE

Echo Envelope Modulation ESEEM

weakly coupled nuclei

(hyperfine & quadrupole)

Field-Swept Pulse EPR

EPR spectra, ns time resolution

FID-detected EPR

t mw

90°

ESE-detected EPR

mw

90°

mw

180°

echo

t t

t

High Frequency / High Field

Peloquin et al,, JACS (2000)

Kulik et al., JACS (2005, 2007)

Exp. Sim.

MnX

50 100 150 200 250

EN

DO

R inte

nsity,

a.u

.

Radiofrequency, MHz

X-Band

Q-Band n

Magnetic field, mT

Simulations:

Exp

4 nuclei all

3 nuclei B,C,D

2 nuclei C,D

1H

1150 1200 1250 1300 1350

EP

R inte

nsity,

a.u

. g = 1.96

g = 2.00

MnA 235 265

MnB 185 245

MnC 310 265

MnD 175 230

A A∥

• There are no 55Mn ENDOR signals above

~180 MHz; all signals fall in the 60-180 MHz range

• MnII not present in the S states

• The spin projection coefficients must all be

approximately 1 (ρ~1) ( see also EPR simulation)

IV

IV

III

IV

JAB

JBC A

B

D

C

JBD JCD

JAC

JAD

width

55Mn ENDOR at X- and Q-Band S2-State of the WOC

Electronic Structure: Multiline EPR and 55Mn ENDOR

O

N

Mn

Ca

D1

A4

B3

C2

Open Coord. Site (H2O/-OH)

III IV

IV

IV

Glu189

Asp342

His332

C2

A4 B3

D1

JAB JCD

JBC

JBD

250 300 350 400

Ca2+

Sr2+

Field (mT) Radio Frequency (MHz)

Data

Sim. (OEC)

Sim. (Mn2+)

100 200 300

1

2

3

4 5 6

1

2

3

4

5 6

1 H

1 H

A B Ca2+

Sr2+

IV

IV

IV III

a

b

b

a

CW

EP

R -

(X

-Band

)

EP

R-d

ete

cte

d N

MR

Electronic Structure Exchange Coupling Scheme

-Ca2+

-Ca2+ 1H 1

2 3

4

5

Oxidation States (S2) Structural Model (S2)

EPR

Pantazis et al. PCCP (2009)

Cox, Rapatskiy et al. JACS (2011)

Ames et al. JACS (2011)

Lohmiller et al. JBC (2012)

S2 State

S2 State

DFT

17O Labelling of PSII

PSII

H217O (90%)

55Mn 40Ca 16O 1H

C2 A4

B3

D1

C

A

B

D

55Mn 40Ca 16O 17O 1H

All oxygens

(i.e. possible

substrates)

EPR/NMR

silent

Exchanged

sites now

EPR/NMR

active

3x exchanged (in dark, S1 state)

1 hour

75% enrichment (final)

1:1

prfoff / pfoff

prfon / pfon

npump

ndetect

p f

B) ELDOR-detected NMR

t

p p / 2 t

y 1

y 2

y 3

y 4

y 1

y 2

y 3

y 4

pf = b14

y 1

y 2

y 3

y 4

p r f

A) Davies ENDOR

t

p p / 2 t

p a

y 1

y 2

y 3

y 4

y 1

y 2

y 3

y 4

pa = b13

prf = b34

ENDOR versus ELDOR-detected NMR (EDNMR)

17O EDNMR vs. ENDOR

W-band (94 GHz)

10

Radio Freq. (MHz)

20 30

MnII(H2O)6

20 min

12 h

PS II S2

n(14N)

n(17O)

Rapatskiy et al., J. Am. Chem. Soc. in press

14N

0 10 20 30 40 50 60

Dn = n mw

- n mw (0) (MHz)

Aiso(μ-oxo) ~10 MHz

Aiso(OH) ~4 MHz typical values seen in models

agrees with DFT calculations

Three 17O species are required to reproduce the entire envelope

1. Weakly coupled water (matrix) – Ca-W1/W3/W4

2. Intermediary coupled water (Mn bound) – MnA4-OH (W2)

3. Strongly coupled water (μ-oxo bridge)

C2 A4

B3

D1

W1

W2

W3 W4

S2 O4

O5

His332 His332

W1,W3,W4

O4 or O5

W2

17O DQ

ED

NM

R a

mp

litu

de

17O SQ

17O ELDOR-detected NMR at 94 GHz - Simulations

W-band

EDNMR

n mw

- n mw

(0) (MHz)

All three 17O species are seen

after dilution in H217O (1x) and

rapid freezing (<15 s)

1. Matrix – Ca-W3/W4 and W1

2. Mn bound – MnA4-OH/OH2

3. μ-oxo bridge –O5

C2 A4

B3

D1

W1

W2

W3 W4

S2 O4

O5

His332

0 10 20 30

ED

NM

R a

mp

litu

de

15 s dilution

PSII-Ca

PSII-Sr

n mw

= 94 GHz

His332

W1,W3,W4

O4 or O5

W2

14N

17O SQ

Rapid Dilution in H217O; the same signal envelope is seen!

O5 + W3

O5 + WX

II. Oxo/oxyl

radial coupling

C2 A4

B3

D1

W1

W2

W3 W4

55Mn 40Ca

16O 1H

C2 A4

B3

D1

W1

W2

W3 W4

C2 A4

B3

D1

W1

W2

W3 W4

S1

S4

Substrate

Possible Mechanism of Water Oxidation

O

MnIV

MnIV

O O MnIV

*or O5 + W2

I: Nucleophilic attack

MnIV = O

or

MnV O

H2O-Ca

or

HO-Ca

C2 A4

B3

D1

W1

W2

W3 W4

O4

O5

His332

C2 A4

B3

D1

W1

W2

W3

W4

O4

O5 His332

100 200 300 400 500 Magnetic Field (mT)

• O5 is a μ-oxo bridge to the Ca (Ca tunes pKa of bound water(s) )

• O5 has a flexible coordination sphere

‘g = 4.1 state’

SG = 5/2

‘multiline

state’

SG = 1/2

S2 S2

~150 K

IR illum.

~200 K

Anneal

Pantazis et al. , Angew. Chem. (2012)

Boussac et al. Biochemistry (1996)

g=4.1 g=2.0

Simulation

X-band hn = 0.3 cm-1

Mn(III)

Mn(III)

Neese

Cooperation

Why is O5 exchangeable on a seconds timescale?

Lessons from Nature: A light-driven water splitting machine

1. Choice of the catalyst: earth-abundant metals (Mn, Ca) with optimized physicochemi-

cal properties and simple (bridging) ligands (O) forming a cage-like/dangler structure

2. Nuclearity of cluster: 4 Mn for storage of oxidizing equivalents – charge accumula-

tion: coupling of one-electron charge separation to four-electron water oxidation

4. Co-metal: Ca for water binding/delivery

6. Substrate water „integration“ in the cluster: active participation of the catalyst

5. Correct and precise sequential substrate water binding in juxtaposition at the metals,

deprotonation and preparation for O-O bond formation

3. Redox leveling at the catalyst: avoiding high-energy intermedites during the reaction

7. Proton coupled electron transfer PCET: stepwise H+ release for charge neutrality

8. Efficient interfacing of ET chain and catalyst via Yz – lowering the overpotential

9. Smart Matrix: correct binding Mn4Ca-cluster, channels for educts & products, proton

management; electrostatic environment, ET and catalyst dynamics, protection -

all that providing high efficiency (TOF) of the catalyst

10. Self assembly of the catalyst; efficient repair “self healing“ in case of damage –

provides good stability and long lifetime

Hydrogenases:

Spectroscopy & Electrochemistry,

Structure, Function and Oxygen Sensitivity

Ogata et al. (2005) Structure v13 pp. 1635-42

Desulfovibrio vulgaris Miyazaki F, PDB 1WUI Desulfovibrio desulfuricans, PDB 1HFE

[FeFe] Hydrogenase [NiFe] Hydrogenase

Volbeda et al., 1995

Higuchi et al., 1997

Peters et al., 1998

Nicolet et al., 1999

FeFe

COCOCNNC

SS

CO

X

S

[4Fe4S]

CysFeNi CN

S COS

S

XS

Cys

CN

Cys

Cys

Cys

[3Fe-4S]

[4Fe-4S]

large

subunit

small subunit

[4Fe-4S]

[NiFe] center

Nicolet et al. (1999) Structure Fold.Des. v7 pp. 13-23

small

subunit

large

subunit

[FeFe] center

[4Fe-4S]

[4Fe-4S]

H2 H- + H+ 2H+ + 2e-

Two Main Classes of Hydrogenases

X (C,O,N?)

[NiFe]-hydrogenases [FeFe]-hydrogenases

H2 oxidation

H2 evolution

H2 sensing

(RH)

Energy

generation

(MBH)

Re-generation

of reducing

equivalents

(SH)

disposal

of reducing

equivalents

H2 evolution

Disposal

of reducing

equivalents

H2 oxidation

(Re-)generation

of reducing

equivalents

H2 2 H+ 2 e- + uptake

evolution

Biological Functions of Hydrogenases

Activity:

102 – 103

[µmol H2/min*

mg protein]

Activity:

103 – 104

[µmol H2/min*

mg protein]

The most active [FeFe] hydrogenases produce

10,000 molecules H2 per second at 30oC

‘... one mole of such a hydrogenase could produce

enough hydrogen to fill the Graf Zeppelin in 10 minutes......’

R. Cammack, Nature (1999)

Hydrogenases: Catalytic Activity

Initial Questions (AC)

• intermediates of the reaction cycle: redox states of active site and ET components.

• Formal oxidation and spin states of the metal ions Electronic configurations of the ground states.

• Identification and function of the diatomic ligands at the Fe and the bridging ligand X.

• Intermediates in the reaction cycle and their geometry

• Binding site of the substrate hydrogen.

• Effect of light on the hydrogenase intermediary states.

• Impact of the protein surrounding.

• Mechanism of enzyme inhibition (e.g. by O2 or CO) and oxygen tolerance.

• Mechanism of activation/deactivation and reversible hydrogen conversion by hydrogenases

• Structural basis for enzyme activity (TOF) and lifetime (TON)






Peters et al., 1998


[3Fe-4S]

[4Fe-4S]

large

subunit

small subunit

[4Fe-4S]

[NiFe] center


small

subunit

large

subunit

[FeFe] center

[4Fe-4S]

[4Fe-4S]

H2 H- + H+ 2H+ + 2e-

[NiFe]- und [FeFe]-Hydrogenasen

H+ H-

H- H+

Active intermediate Structure of the active

(hydrogen-carrying) intermediate

Distance between Ni and H- is

d = (1.7 ± 0.1) Å and d = (1.8 ± 0.1) Å

between Fe and H-

Ni-C (H/D exchange)

Brecht et al., JACS 2003

Hyperfine Spectroscopy: ENDOR & ESEEM

[3Fe-4S]

[4Fe-4S]

[NiFe] center

large

subunit

small subunit

H+ + H- ⇌ H2

[4Fe-4S]

CN

CN

CO

active

center

x

Fe Ni

Hydrogenase Structure

MW

RF

p

p p

p p/2

ptr

14 16 18 20 22 24 26

RF [MHz]

T = 10K

H2O / H2

D2O / D2

nH

Structure of the

active intermediate

H-

Fe Ni

a

(2D-Puls EPR) HYSCORE

Frequency n1 [MHz]

Fre

qu

en

cy n

2 [

MH

z]

gx gy gz

Brecht et al. JACS (2003)

EPR

4-Pulse ESEEM

gz

gz

Ni-C

Ni-L

EPR and HYSCORE Spectra of Ni-C and Ni-L H/D Exchanged NiFe-Hydrogenase RH of R. eutropha

-8 -6 -4 -2 0 2 4 6 8

Pulse Q-band 1H ENDOR position gy

T = 10 K

Ni-L2

Regulatory Hase

nENDOR - nfree [MHz]

H2O/H2, hn

D2O/D2, hn

Difference

AH ex

Flores et al. unpublished

Ni Fe

H -

3.1 Å

S (Cys81)

S (Cys546)

ENDOR of Ni-L: H/D Exchanged, RH of R. eutropha

Mechanistic insights:

L. De Gioia, P. Fantucci, B. Guigliarelli, P. Bertrand,Inorg. Chem. 1999, 38.

Rauchfuss et al. 2009

Shafaat et al. 2012

Hydrogen Conversion Mechanism of [NiFe]-Hydrogenases

Weber et al. 2012, JACS

10.8 Å

[4Fe4S]

[4Fe4S]

[3Fe4S]

Proximal [4Fe4S]

e-

H+

H2

[NiFe]

FeII NiII H2O

e-, H+

FeII [O] NiIII

FeII [O] NiII FeII(OH-)NiII

FeII(OH-)NiIII

Ni-A

Ni-SU

Ni-B

(Ni-SIr)I

+H+

FeII NiII

FeII(H-)NiIII

FeII(H-)NiII

Ni-SIa

Ni-C

Ni-R(1,2,3)

e-, H+

-H+

e-, H+

e-, H+

(Ni-SIr)II

active

states

inactive

states

FeII NiII(OH-)

Ni-SL

light-induced

states

hn

+H2O

-H2O ) (

Ni-SCO

FeIINiII(CO)

Ni-CO

FeIINiI(CO) - CO

+ CO

CO-inhibited

states

+ CO

- CO

light-induced

states

FeII NiI hn / -H+

+H+

Ni-L(1,2,3)

Reaction Scheme for Standard [NiFe] Hydrogenases

Medial [3Fe4S]

Distal [4Fe4S] 9.4 Å

8.5 Å

Pandelia, Ogata, Lubitz,

ChemPhysChem, 11,1127 (2010)






Peters et al., 1998


[3Fe-4S]

[4Fe-4S]

large

subunit

small subunit

[4Fe-4S]

[NiFe] center


small

subunit

large

subunit

[FeFe] center

[4Fe-4S]

[4Fe-4S]

H2 H- + H+ 2H+ + 2e-

X

H

N

H+ H-

H+

[NiFe]- und [FeFe]-Hydrogenasen

H.-J. Fan, M.B. Hall, J. Am. Chem. Soc. 123 (2001) 3828.

Z.-P. Liu, P. Hu, J. Am. Chem. Soc. 124 (2002) 5175.

Hox: Fe(I)pFe(II)d

Hred: Fe(I)pFe(I)d

S S S

Fe Fe

NC

OC CO

CN C O

N

Cys

[4Fe4S]H

H

H(-)

H(+)

S S S

Fe Fe

NC

OC CO

CN C O

N

Cys

[4Fe4S]H

H

H

H

S S

Fe Fe

N

14N in the dithiolate bridge: Implications for the H2 splitting mechanism

H

14N detected via pulse EPR

- exp - N1 - N2 - N3

14N HYSCORE Silakov et al.

PCCP (2009)

active site

model compound

native system Hox

active site

AN (x, y, z) [MHz] (0.3, 0.8, 0.7) (1.0, 1.9, 1.4)

Aiso [MHz] 0.6 1.43

K [MHz] 1.28 1.23

η 0.11 0.13

S S S

PMe3 PMe3

14N

CH3 Fe Fe

OC

OC CO

C

O

Frequency [MHz]

Fre

quency [M

Hz]

Fre

quency [M

Hz]

14N 15N

14N 15N

exp

Frequency [MHz]

2 4 6 0 0

2

4

6

2 4 6 0 0

2

4

6

2 4 6 0 0

2

4

6

2 4 6 0 0

2

4

6

exp

sim sim

Ö Erdem, et al.

Angew. Chem. Int. Ed.

50, 1439 (2011)

[2Fe3S](PMe3)2 complex, 14N & 15N HYSCORE

15N

+

Cooperation S. Ott (Uppsala)

[FeFe] Hydrogenase: Proposed Catalytic Mechanism

Hox

Hred

- - e

Hsred

+H2

+e

+e -

- H2

+H+

- H+

-

-e -

- H+ +H+

FeI FeII [ H2 ] 2+

S S

N H

C

O

2+ FeI FeII [ ]

S S

N H

C

O

2+

S S

N H

H +

FeI FeI [ ]

C

O

FeI FeII [ H- ] 2+

S S

N H

H +

C

O

FeI FeI [ ] +

{H+} S S

N H

C

O

2+ FeI FeII [CO]

S S

N H

C

O

+ CO

- CO

Hox-CO

Cooperation T. Happe (Bochum) A. Adamska et al.

Angew. Chem. Int. Ed. (2012)

Bronsted basic

bridging atom, nitrogen

Lewis acidic

metal center

electron supply/

asymmetry

Essential components of the hydrogen producing active

site of [FeFe] hydrogenase:

Reaction: H2 H- + H+ 2H+ + 2e-

A. Metal center at optimal potential and spin state (CN/CO ligands) to

polarize H2 for heterolytic splitting (open coordination site!) and accept

hydride as ligand (hydride acceptor ability)

B. Adjacent base to accept proton (H+ acceptor ability) in concert with

metal

C. Energetic matching of metal and base acceptor reactions to ensure

reversibility, avoid high energy intermediates and thereby achieve higher

rates and lower overpotentials for both reactions

D. Provide efficient delivery of educts and transport of products (solvent;

water; smart matrix) and efficient, optimized electron transport chain

E. Solve problem of oxygen sensitivity

F. Solve problem of degradation (self-repair, self-healing, simplicity)

F. Integrate into a device for (sun) light harvesting, charge

separation/transport, water oxidation catalysis

– including a membrane system (H+, e-/h+ movement)

Design criteria for artificial hydrogenase catalysts:

Science, 2011,333, 863

Angew. Chem. Int. Ed. 2012, 51, 3152

Some Unsolved Problems:

1. Oxygen sensitivity of catalysts

2. Self-assembly & self-repair - long term stability

3. Fast rates TOF

4. Low overpotential

5. Interfacing (e.g. to electrodes) – development of linkers

6. Combination with water oxidizing catalysis

7. Combination with (sun) light induced processes

(antenna, charge separation etc.)

8. Costs – materials, difficulty of synthesis

0 1000 2000 3000 4000

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Curr

en

t (µ

A)

Time (s)

[O2] = 15 µM

[NiFe] Hydrogenase D. vulgaris MF

H2

2H+

Fe

Ni e-

e- Graphite

Fe-S

Fe-S

Fe-S

H2 H2

H2

H2

H2 H2

O2

O2

O2

O2

O2

Protein Film Electrochemistry

PFE

E=+150 mV vs NHE

40ºC, pH 6.0

2500 rpm, 1 bar H2

E=+150 mV vs NHE

40ºC, pH 6.0

2500 rpm, 1 bar H2

1000 2000 3000 4000 5000

20

40

60

80

100

120

140

% R

ecovery

Time (s)

[O2] = 15 µM

[O2] = 59 µM

[O2] = 108 µM

[O2] = 205 µM

(sat at 40°C)

[NiFe] Hydrogenase A. aeolicus

Oxygen Inhibition of Hydrogenases

1. Narrower gas access channel

2. Blockage of active site by

additional ligands (CN-)

3. Modification of redox potentials

of the active site and the electron

transport components

(proximal 4Fe4S-cluster)

[3Fe-4S]

[4Fe-4S]

[NiFe] center

large

subunit

small subunit

CN

CN

CO

active

center

x

[4Fe-4S]

Fe Ni

Oxygen Tolerance of [NiFe] Hydrogenases

1 e- 2 e- (!) 1 e-

4 distinct EPR species 1 2 3 4

Redo

x p

ote

ntial, m

V v

s N

HE

[NiFe] [Fe4S4] [Fe4S4] [Fe3S4]

+290

0

-180

S(1/2)

S(1/2)

S(1/2)

+120 S(1/2)

1+

1+ 1+

2+

2+

2+

2+

1+

1+

0

0

Pandelia et al.,

PNAS 2011

EPR Redox Titration of the FeS Clusters in A. aeolicus

Proximal

4Fe4S-cluster

in A. aeolicus

[Fe4S4]

distal

8.5 Å Electron transfer chain

in standard enzymes

[Fe3S4]

medial

[Fe4S4]

proximal

10.8 Å

9.5 Å

[NiFe]

site

Cys19

Cys17

Cys149

Cys120

Cys115

Cys20

A.vinosum T.roseopersicina D.vulgaris D.gigas D.desulfuricans D.fructosovorans D.baculatum A.aeolicus R.eutropha MBH

A.vinosum T.roseopersicina D.vulgaris D.gigas D.desulfuricans D.fructosovorans D.baculatum A.aeolicus R.eutropha MBH

Sequence Alignment Showing Binding of the Proximal FeS Cluster

reduced

superoxidized

+2

+3

+2.5

+2

+2.5

+3

+2.5

+2.5

+2.5

+2.5 +2.5

Stot = 1/2

Stot = 1/2

oxidized Stot = 0

+2.5

Proximal [4Fe-3S] Cluster: Mössbauer Analysis

Pandelia et al. (2013)

PNAS

H2O

O2

+ 3H+

H+, e-

H2

H+ e-

(Base)

H+

H2O

e-

rapid

activation

NiIII FeII

S S

H-

NiIII FeII

S S

OH-

NiII FeII

S S

Hydrogenase Oxygen Reductase

Oxidase

[3Fe4S]

[4Fe3S] prox

[4Fe4S]

+ 3e-

Hydrogenase Catalysis in the Presence of Oxygen

Proximal cluster

[4Fe-3S]-6Cys

Cracknell et al. PNAS (2009); Goris et al. Nature Chem. Biol. (2011)

reduced (super)oxidized

Wenk

Kellers Ogata Stein Flores Pandelia

Silakov

van Gastel

Erdem Reijerse

Acknowledgements

[FeFe] Hydrogenases and related model systems:

[NiFe] Hydrogenases and related model systems:

Christof Geßner

Marc Brecht

Stefanie Foerster

Olga Trofanchuk

Kamp

Fichtner

Hydrogenases

Rüdiger

Support:

European Union NEST: SOLAR-H2, Alexander von Humboldt Foundation, Max Planck Society

Cooperations:

Y. Higuchi (Hyogo)

B. Friedrich (Berlin)

S. Albracht (Amsterdam)

MT. Giudichi-Orticoni

(Marseille)

T. Rauchfuss (Urbana, Ill.)

Shafaat Nishikawa Weber Bill

Support:

European Union NEST: SOLAR-H2, Alexander von Humboldt Foundation, Max Planck Society

Acknowledgements Wateroxidase

Ogata Sander

Kulik Epel Su Rapatskiy Lohmiller

Orio Pantazis Krewald Ames

Wieghardt Weyhermüller Messinger

(Umeå)

Boussac

(Saclay)

Spectroscopy

Theory

Cooperations

Crystallography & Preparations

Rögner

(Bochum)

Neese

Cox Pérez Navarro

Krause

Nowaczyk

(Bochum)

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